Beneficial effects of bacteriophage treatments

ABSTRACT

The invention relates to use of one or more bacteriophages in vivo in a human or animal in order to induce sensitivity to chemical antibiotics in bacterial cells, where such susceptibility is heritable, independent of continuing bacteriophage metabolism within those cells, and does not relate to the destruction of a biofilm to induce such sensitivity.

FIELD OF THE INVENTION

The present invention relates to the sensitisation of previouslyresistant bacteria to antibiotics following treatment withbacteriophages. In particular, the invention provides in its preferredaspect for the preparation and administration of therapeutic medicamentsthat use sequential treatments with bacteriophages and conventionalantibiotics, to be used with infections of animals and humans caused bypathogenic bacteria.

BACKGROUND TO THE INVENTION

Antibiotic resistance is now seen as one of the major challenges facingmodern medicine. Given the shortage of novel antibiotics, a number ofalternative approaches are being investigated, including the use ofbacteriophages as therapeutic agents (Barrow & Soothill, Trends inMicrobiology (1997), 5, 268-271; Dixon B, The Lancet Infectious Diseases(2004), 4, 186; Hausler T, Viruses vs. Superbugs: A Solution to theAntibiotics Crisis? (2006) MacMillan, New York; Matsuzaki et al, Journalof Infection and Chemotherapy (2005), 11, 211-219.

Bacteriophages (often known simply as “phages”) are viruses that growwithin bacteria. The name translates as “eaters of bacteria” andreflects the fact that as they grow most bacteriophages kill thebacterial host as the next generation of bacteriophages is released.Early work with bacteriophages was hindered by many factors, one ofwhich was the widespread belief that there was only one type ofbacteriophage, a non-specific virus that killed all bacteria. In fact,the host range of bacteriophages (the spectrum of bacteria they arecapable of infecting) is often very specific. This specificity may beconsidered a therapeutic strength as populations of bacteriophages canbe selected to specifically eliminate only the target bacterial species.

Despite the therapeutic advantages afforded by the host specificity ofbacteriophages, this characteristic has the disadvantage that it can bedifficult to achieve breadth of coverage of target strains. For thisreason, there has been interest in finding combinations ofbacteriophages having broad targeting capability in relation toparticular types of bacterial infection (see for example Pirsi, TheLancet (2000) 355, 1418). This has now been achieved with thedevelopment of a mixture of six bacteriophages targeting Pseudomonasaeruginosa, which has completed veterinary field trials and is now inhuman clinical trials (Soothill et al, Lancet Infectious Diseases (2004)4, 544-545). The challenge now is to develop regimens which optimise thedelivery of such therapies.

Bacteriophages and antibiotic therapy have previously been used togetherin Eastern Europe (see for example Bradbury, The Lancet (February 2004)363, 624-625), but without specific reporting of synergistic effects.Indeed, there have been suggestions that antibiotics can have adverseeffects on use of bacteriophage therapy since bacteriophages usebacterial metabolism to replicate and this is inhibited by antibiotics(Payne and Janssen, Clinical Pharmacokinetics (2002) 42, 315-325).

More recently, bacteriophages have been shown to produce benefits wheremixed pathogenic bacteria grow in a biofilm (Soothill et al, 2005, PCTpatent application WO2005009451). In this application benefit was shownwith respect to subsequent antibiotic treatment of heterologousbacterial infections, apparently by disruption of the biofilm followingbacteriophage treatment.

Biofilm formation is now known to be a characteristic of many importantpathogenic bacteria contributing to increased resistance to antibiotics.Such biofilms may comprise more than one type of bacterium supported andsurrounded by an excreted extracellular matrix and assist bacteria tocolonise surfaces from marine reefs to tooth enamel. Biofilms allowbacteria to attach to surfaces and to attain population densities whichwould otherwise be unsupportable. They impart increased resistance tonot only antibiotics but many environmental stresses including toxinssuch as heavy metals, bleaches and other cleaning agents. It waspreviously thought that contribution of biofilm formation to antibioticresistance was primarily a physical process arising from limitation ofdiffusion, but more recent evidence has shown that some biofilms appearto have specific abilities to trap antibiotics (Mah et al., Nature(2003) 426, 306-310). It is known that bacteria within biofilms can be100 to 1000 times more resistant to antibiotics than the same strain ofbacteria growing in single-celled (“planktonic”) forms. This increasedresistance means that bacteria that are apparently sensitive toantibiotics in a laboratory test may be resistant to therapy in aclinical setting. Even if some are cleared, biofilms may provideresistant reservoirs permitting rapid colonisation once antibiotics areno longer present. It is clear therefore that biofilms are major factorsin many human diseases.

As noted above, greater beneficial effects have been observed with thesubsequent use of antibiotics against mixed infections following the useof a therapeutic bacteriophage preparation against Pseudomonasaeruginosa, and it has been proposed that this is due to the destructionof Pseudomonas aeruginosa as the key species maintaining the biofilm(Soothill et al, 2005, PCT patent application WO2005009451), whichresults in loss of biofilm integrity and thus exposure of bacteria toconventional antibiotics.

The teaching of PCT patent application WO2005009451 is against the useof antibiotics that are specifically active against the same bacterialspecies as that targeted by bacteriophage. The examples cited refer tothe use of Synulox (amoxicillin and clavulanic acid) and/or Canaural eardrops (containing diethanolamine fusidate, framycetin sulphate, nystatinand prednisolone). Both of these preparations contain only antibioticsthat are not effective against Pseudomonas aeruginosa (Krogh et al,Nordisk Veterinaer Medicin (1975) 27, 285-295; Kucers A, in Kucers et al(eds), The Use of Antibiotics: A Clinical Review of Antibacterial,Antifungal and Antiviral Drugs, Fifth edition (1997),Butterworth-Heinemann, Oxford; Rawal, Journal of AntimicrobialChemotherapy (1987) 20, 537-540). In particular, while aminoglycosideantibiotics as a class are effective against Pseudomonas aeruginosa,Framycetin is of very limited efficacy. Kucers notes that “Nearly allthe medically important Gram-negative aerobic bacteria are sensitive”(to Neomycin, Framycetin and Paromomycin) “with the exception ofPseudomonas aeruginosa”, while the same author states that “Pseudomonasaeruginosa is co-amoxiclav resistant, citing the work of Comber et al,in Rolinson & Watson Augmentin (eds) (1980), Excerpta Medica, Amsterdam,p.19. Co-amoxiclav is defined in the online 52^(nd) edition of theBritish National Formulary (www.bnf.org) as “a mixture of amoxicillin(as the trihydrate or as the sodium salt) and clavulanic acid (aspotassium clavulanate), equating to the veterinary drug Synulox. Thus,PCT patent application WO2005009451 would not indicate the use ofantibiotics targeting Pseudomonas in any combination with bacteriophagesbut rather the use of antibiotics specifically targeting co-infectingbacteria.

Another mechanism has been identified recently by which bacteriophagescan increase the sensitivity of bacteria to antibiotics to which theyare resistant (Hagens et al, Microbial Drug Resistance (2006), 12,164-168). This involves active bacteriophage metabolism, and issuggested to involve the formation of pores in the bacterial membrane.However, this teaches that “resensitization of pathogens resistant to aparticular antibiotic can be achieved in the presence of phage in vivo”based around the use of “a combination treatment with antibiotics andfilamentous phage”. Thus this relates to a non-heritable characteristicwhich is exerted only in the presence of bacteriophage, which relies onthe simultaneous use of both bacteriophages and antibiotics, and whichappears to be specific to filamentous to bacteriophages which form poresin the bacterial membrane. This is thus distinct from the inventionsclaimed herein, which induce heritable changes that persist even whenactively replicating bacteriophage is not present.

SUMMARY OF THE INVENTION

The present invention is based on the induction of sensitivity tochemical antibiotics by the use of bacteriophage treatment in vivo inhumans or in animals, where such sensitivity is heritable, does not relyon active bacteriophage metabolism and does not relate to thedestruction of biofilm to induce such sensitivity, along with thepreparation of medicaments to permit the sequential use ofbacteriophages and antibiotics so as to take advantage of such inductionin sensitivity in the control of bacterial disease, especially forexample a Pseudomonas aeruginosa infection. Induction of sensitivity inthis context will be understood to include improvement of sensivity.

DETAILED DESCRIPTION

Thus in one aspect, there is provided a bacteriophage preparationcomprising one or more bacteriophages for use in combined bacteriophageand antibiotic therapy to treat a bacterial infection in a human oranimal, wherein at least one antibiotic is administered following thestart of said bacteriophage treatment at a time period at whichsusceptibility of bacterial cells of said infection to said antibioticis induced or improved by the bacteriophage treatment, where suchsusceptibility is heritable, independent of continuing bacteriophagemetabolism within those cells, and does not relate to the destruction ofa biofilm to induce such sensitivity. Antibiotic sensitivity may bemonitored by established procedures in vitro. Induction of sensitivitymay be confirmed for one or more bacterial strains from the individualpatient or may be identified in other patients with similar bacterialinfections following bacteriophage treatment

In a further aspect, there is provided a two stage medicament where thefirst stage comprises a bacteriophage-based therapeutic and the secondis composed of one or more chemical antibiotics, for sequential use inhumans or animals, where this is designed to exert beneficial effects bythe induction of sensitivity as noted above.

The bacteriophage therapeutic and one or more chemical antibiotics maybe administered for example at an interval of one to two days to twomonths apart, preferably at an interval of one to four weeks, mostpreferably at an interval of two weeks apart.

As indicated above, combined phage/ antibiotic therapy according to theinvention may be particularly useful for example in targeting bacterialinfection comprising or consisting of Pseudomonas aeruginosa. Suchinfection may be, for example, at the site of a skin burn or other skinwound. It may be in the lung, an ocular infection or an ear infection.In this context, such an infection comprising P. aeruginosa will beunderstood to include an infection consisting essentially of P.aeruginosa. Thus, phage therapy according to the invention may beapplied to an infection composed entirely, predominantly orsignificantly of P. aeruginosa.

EXAMPLES

Induction of Antibiotic Sensitivity in a Veterinary Field Trial:

Canine ear infections caused by Pseudomonas aeruginosa (otitis externaand otitis media) are examples of clinical disease associated withbiofilm-based colonization of a body surface. Clinical signs of suchinfection include pain, irritation (erythema), ulceration and thedischarge of increased amounts of material from the ear. This is oftenpurulent in nature and is accompanied by a distinctive odour.

A combined preparation of six bacteriophages was named BioVet-PA, andwas authorized for trial in dogs with such infection by the VeterinaryMedicines Directorate of the United Kingdom in November 2003.

Conduct of the Trial

BioVet-PA was stored at −80° C. Immediately prior to administration, theproduct was thawed and warmed in the hand. 0.2 ml (containing 1×10⁵infectious units of each of the 6 bacteriophages) was administereddrop-wise using a sterile 1 ml capacity syringe into the ear. Earcondition and microbiology was assessed at 2 days post-administration.

The procedure was as follows:

Characterisation (2 to 14 days prior to treatment):

Day 0 Swabs taken from each ear by a veterinary surgeon Laboratory testscarried out using these swabs to confirm presence of Pseudomonas.aeruginosa.

If Pseudomonas aeruginosa was not detected, the dog was excluded fromthe trial

Day 1 If Pseudomonas aeruginosa was detected, the isolates were testedfor sensitivity to BioVet-PA.

If the Pseudomonas aeruginosa strain(s) with which the dog was infectedwas/were not sensitive to BioVet-PA, the dog was excluded from thetrial.

Treatment:

Day 0 Ears examined auroscopically to assess their condition. Swabstaken from each ear for microbiological analysis. Dog's core temperaturemeasured Dog given dose of 0.2 ml BioVet-PA into the ear (treatmentsadministered drop-wise using a sterile 1 ml-capacity syringe, and earcanals then massaged to promote deep penetration). Day 2 Ears examinedto assess their condition. Swabs taken from each ear for microbiologicalanalysis. Dog's core temperature measured. Only where both ears wereinfected: Dog given dose of 0.2 ml BioVet-PA into the second ear(treatments administered drop-wise using a sterile 1 ml-capacitysyringe, and ear canals then massaged to promote deep penetration). Day4 Only where both ears were infected: Ears examined to assess theircondition. Swabs taken from each ear for microbiological analysis. Dog'score temperature measured.

RESULTS

Studies on ten dogs with severe, antibiotic-resistant Pseudomonasaeruginosa ear infections treated with BioVet-PA showed improvement inclinical symptoms within two days of treatment and reductions inbacterial numbers over the same timescale. Bacteriophage replication wasobserved in all dogs. Analysis of the improvement in clinical symptomsshowed this to be significant at the 95% level of confidence by both thet-test and the Wilcoxon matched-pairs test.

Three dogs were excluded from the trial. The first dog to be tested wasexcluded because of a subsequent change in the scoring system (to takeaccount of ear discharge purulence) while two treated dogs proved tohave infections that were not predominantly with the target bacterium atthe time of treatment (due to a change in bacterial flora followingpre-entry screening).

Antibiotic Resistance:

All isolates of Pseudomonas aeruginosa from all thirteen dogs that werecollected before treatment were screened for their sensitivity toantibiotics. The antibiotic sensitivity profile of each Pseudomonasaeruginosa strain was assessed with a range of 10 antibiotics which maybe used clinically by veterinarians to treat Pseudomonas aeruginosainfections. The results were recorded in appropriate data collectionsheets.

Since differing colony types were often observed in the same dog andboth ears were infected in four dogs, a total of 83 individual isolatesof Pseudomonas aeruginosa were tested. Thus, 830 tests were carried out,of which 340 were on swabs taken immediately prior to treatment, 340 twodays after treatment, and 150 four days after treatment.

All sensitivity assays were compared to identify shifts in sensitivityto any of the antibiotics tested. No individual isolate showed more thana single shift, and no isolate changed from fully sensitive to fullyresistant, or fully resistant to fully sensitive. However, shifts fromsensitive to partially resistant, partially resistant to resistant,resistant to partially resistant, or partially resistant to sensitivewere seen for 16 isolates. Events were observed as shown in Table 1below.

TABLE 1 Piperacillin + Meropenem Tazobactam Colistin Imipenem AmikacinCeftazidime Ciprofloxacin Gentamicin Aztreonam Tobramycin All sensitiveSensitive 1 change to Partially resistant Partially 1 resistant changeto Resistant Resistant 6 3 5 change to Partially resistant Partially 2 25 5 resistant change to Sensitive Monitored isolates in total 340  100%more resistant  2 0.59% more sensitive  28 8.24%

A total of 30 alterations in antibiotic sensitivity were seen, with 28being shifts towards sensitivity and 2 being shifts towards resistance.Thus shifts towards sensitivity outnumbered those towards resistance bya factor of 14:1, illustrating the predominance of such “beneficial”shifts following bacteriophage treatment.

Induction of Antibiotic Sensitivity in a Human Clinical Trial

The trial was a single-centre, double-blind, randomised, parallel groupstudy of the safety and efficacy of a single administration ofBioPhage-PA (a mixture of six bacteriophages specific for Pseudomonasaeruginosa) compared with placebo in patients with chronic ear infectioncaused by Pseudomonas aeruginosa shown to be susceptible to one or moreof the bacteriophages present in BioPhage-PA.

This study investigated the efficacy and safety of BioPhage-PA, amixture of six Pseudomonas aeruginosa bacteriophages of the same typesas those tested as BioVet-PA in the veterinary field trial, which formedpart of the pre-clinical work for this study.

The six bacteriophage strains (which were deposited at the NationalCollection of Industrial and Marine Bacteria, 23 St Machar Drive,Aberdeen, AB24 3RY, Scotland, UK on 24 Jun. 2003) are as follows:

Reference NCIMB Deposit Number BC-BP-01 NCIMB 41174 BC-BP-02 NCIMB 41175BC-BP-03 NCIMB 41176 BC-BP-04 NCIMB 41177 BC-BP-05 NCIMB 41178 BC-BP-06NCIMB 41179

These bacteriophages are effective at killing a broad range of P.aeruginosa isolates.

The study was carried out in two parallel groups of patients with earinfection caused by to Pseudomonas aeruginosa. Patients were randomlyallocated to receive a single dose of either BioPhage-PA or placebo andwere be monitored in a double-blind design over a period of 6 weekspost-dose. Efficacy assessments included questions about adverse events,both patient and investigator assessment of disease severity usingvisual analogue scales, Pseudomonas aeruginosa and bacteriophage earswab count, audiogram, photography of the ear, and aural temperatureanalysis. Change from baseline (pre-dose assessment) in active andplacebo groups were compared statistically. Safety data was alsocompared in the 2 groups.

Study Design

This was a single-centre, double-blind, randomised, parallel group studyin patients with chronic Pseudomonas aeruginosa infection of the ear.Patients were randomised to one of two groups:

Group 1:

Patients received a single 0.2 mL dose of BioPhage-PA (containing 1×10⁵pfu by original titration of each of the 6 therapeutic bacteriophages)

Group 2:

Patients received a single 0.2 mL dose of placebo (10% v/v glycerol inPBS)

Design Summary

Pre-Study Visit

Patients attended the clinic within 2 weeks of treatment Day 0 afterbeing informed of the trial verbally. At this visit, they were providedwith a written information sheet and were also provided with studydetails verbally. Patients were questioned regarding their eligibilityto participate and if successful signed a consent form prior to Day 0 ofthe trial.

Treatment Period (Days 0-42 Inclusive)

Patients attended the clinic on the morning of Day 0 for clinicalexamination and were questioned about adverse events and studycompliance. Upon confirmation of eligibility, patients were randomisedto one of the two treatment groups and had baseline assessmentsperformed to determine the severity of the infection. Treatment was thenadministered by the clinician who instilled the therapy drop-wise intothe ear. Patients remained in the clinic for 6 hours post-dose. Theywere issued with diary cards for recording any adverse events orcomments on the condition of the ear on a daily basis whilst away fromthe unit.

Patients returned on Days 7, 21 and 42 for further safety and efficacytests.

A patient was eligible for inclusion in this study only if all of thefollowing criteria apply: Aged 18 or over; able and willing to givewritten informed consent to take part in the study; infection of a theear shown to be caused predominantly or solely by Pseudomonasaeruginosa; Pseudomonas aeruginosa isolated from the infection and shownto be vulnerable to one or more of the bacteriophages present inBioPhage-PA; infection established for at least 6 weeks and provenunresponsive to conventional anti-bacterial therapy; available to attendall clinic visits and complete all study measurements; female patientsto be post-menopausal, surgically sterile or willing to use anacceptable form of contraception.

A patient was not eligible for inclusion in this study if any of thefollowing criteria applied: Local surgery within 3 months of thepre-study visit; acute or systemic sepsis; use of systemic or topicalantibiotics within one week of the pre-study visit or during the study;use of topical antiseptic or anti-inflammatory agents within one week ofthe pre-study visit or during the study; bacteriophage therapy in the 6months prior to the pre-study visit; haemolytic Streptococci of groupsA, B, C and G or unusual bacterial or fungal flora on ear swab cultureat the pre-study visit; female pregnant or intending to become pregnant;patients who have a past or present disease which, as judged by theinvestigator, may affect the outcome of the study; any other conditionwhich the investigator feels may prejudice the results of the study;participation in another clinical trial involving a new molecular entitywithin the previous 4 months or any trial within the previous one month.

Study Assessments and Procedures

Each patient attended the unit for the following visits:

Prior to the official start of the study, swabs (in transport media)were taken from the ears of potential trial candidates. Generalmicrobiological analysis was carried out to determine the level ofPseudomonas aeruginosa in the ear. This was followed by a diagnosticear-swab test. The trial was discussed verbally with patient, and thepatient was provided with information sheet/consent form; history wastaken and recorded on the case report form; a diagnostic swab was takenand the swab sent for microbiological analysis, where it was analysedfor Pseudomonas aeruginosa and for sensitivity of Pseudomonas aeruginosathat was present to the bacteriophages in BioPhage-PA.

If suitable, the patient was enrolled onto trial within 2 weeks of thetime that the diagnostic swab was taken.

Study Day 0: The patient assessed the condition of their ear for:discomfort, itchiness, wetness, and smell. Using pre-weighed dry swabs,samples were taken for microbiological analysis. Oral and auraltemperature were recorded, the ear was cleaned, and the attendingphysician assessed the ear for: erythema/inflammation,ulceration/granulation/polyps, discharge type(clear/mucoid/mucopurulent), discharge quantity, and odour (immediatelyprior to study procedures). Digital otoscopic photography was performed,along with a hearing test (audiogram). BioPhage-PA (0.2 ml) was thenadministered directly into the ear canal using a 1 ml syringe and softsterile tubing over a period of approximately 30 seconds. The patientremained at the clinic for 6 hours after therapy for observation and wasthen sent home with a diary card to record any information they feltrelevant to their condition.

Study Day 7: This involved adverse event and compliance questioning,patient assessment of the ear, swab sampling with microbiologicalanalysis, recording of aural and oral temperature, physician assessmentof the ear, and ear cleaning.

Study Day 21: This involved procedures as described for study day 7

Study Day 42: This involved procedures as described for study days 7 and21, except that a hearing test was also be performed and the ear wasphotographed.

Microbiological Assessment

Microbiological assessment involved counting of the Pseudomonasaeruginosa present on the swab, along with counting of allbacteriophages (both extraneous and therapeutic) on the swab.

Sensitivity to ten antibiotics (as for the veterinary field trial) wasalso monitored for all isolates. The antibiotic sensitivity test wasconducted on each strain of Pseudomonas aeruginosa isolated. The testwas conducted according to the standard methods of the British Societyfor Antimicrobial Chemotherapy (BSAC) Disc Diffusion Method forAntimicrobial Susceptibility Testing (May 2003). The antibiotics to beused were as follows:

-   Amikacin—30 μg/ml-   Ceftazadime—30 μg/ml-   Ciprofloxacin—5 μg/ml-   Gentamicin—10 μg/ml-   Meropenem—10 μg/ml-   Pipericillin+Tazobactam (7.5:1)—85 μg/ml-   Colistin—25 μg/ml-   Aztreonam—30 μg/ml-   Imipenem—10 μg/ml-   Tobramycin—10 μg/ml

The test was conducted using Isosensitest agar.

It was found that in the first patient in which bacteriophagereplication was seen, there was evidence of a movement towardssensitivity for three of the ten antibiotics monitored (see Table 2).

TABLE 2 Antibiotic sensitivity of Pseudomonas aeruginosa: Data fromhuman otitis trial Day 0 Antibiotic Pre-treatment screening (treatment)Day 7 Day 21 Day 42 Amikacin Partially resistant Partially resistantSensitive Partially resistant Sensitive Gentamicin Resistant ResistantResistant Resistant Partially resistant Tobramycin Resistant SensitiveResistant Partially resistant Resistant Meropenem Sensitive SensitiveSensitive Sensitive Sensitive Imipenem Sensitive Sensitive SensitiveSensitive Sensitive Ceftazadime Sensitive Sensitive Sensitive SensitiveSensitive Pipericillin & Tazobactam Sensitive Sensitive SensitiveSensitive Sensitive Colistin Sensitive Sensitive Sensitive SensitiveSensitive Aztreonam Sensitive Sensitive Sensitive Sensitive SensitiveCiprofloxacin Sensitive Sensitive Sensitive Sensitive Sensitive

SUMMARY OF THE ABOVE EXEMPLIFICATION

In the veterinary field trial, over a two to four day monitoring period,evidence was found of a movement towards antibiotic sensitivity in 8.24%of Pseudomonas aeruginosa isolates (against 0.59% where movement towardsresistance was seen).

In human trial, evidence was seen of a movement towards sensitivity tochemical antibiotics following the use of a bacteriophage therapeutic inthe first patient where bacteriophage replication was observed. Suchmovement was seen for three of ten antibiotics monitored (30%), over thelonger monitoring period in this trial.

Further Human Trial Results

Subsequent analysis of all twenty four participants in the human trialconfirmed the above findings as follows with reference to Tables 3 and 4below. It can be seen that cessation of antibiotic treatment (requiredfor trial entry) itself produced a drift towards antibiotic sensitivity,but that this was more marked for both numbers of patients and forindividual antibiotics assayed in the test (bacteriophage-treated)group, with the majority of patients (7/12) showing at least one changetowards sensitivity during the monitoring period. Shifts to sensitivityappear particularly marked for aminoglycoside antibiotics with, forexample, five of twelve bacteriophage-treated patients showing increasedsensitivity to gentamicin. Taken together, the above data exemplifiesthe present invention.

TABLE 3 Antibiotic sensitivity of Pseudomonas aeruginosa: Data fromhuman otitis trial Change from pre-screening to day 42 Placebo groupPatient number Antibiotic 3 4 6 8 9 11 13 14 19 20 22 23 Amikacin + +Gentamicin + − + Tobramycin + + Meropenem Imipenem CeftazadimePipericillin & Tazobactam Colistin Aztreonam + Ciprofloxacin Test groupPatient number Antibiotic 1 2 5 7 10 12 15 16 17 18 21 24 Amikacin +− + + Gentamicin + + + + + Tobramycin − − + Meropenem ImipenemCeftazadime Pipericillin & Tazobactam Colistin Aztreonam + Ciprofloxacin+

TABLE 4 Changes in Antibiotic (ten drugs tested) resistance patterns: Toresistance To sensitivity Test n = No. % No. % Patients (one or more of12 2 16.7% 7 58.3% changes) Individual tests of 120 3 2.5% 11 9.2%Control Patients (one or more of 12 1 8.3% 5 41.7% changes) Individualtests of 120 1 0.8% 7 5.8%

1. A method to induce sensitivity to chemical antibiotics in bacterialcells, comprising administering one or more bacteriophages in vivo in ahuman or animal; and monitoring induction of the sensitivity byantibiotic sensitivity testing in vitro, both before and after theadministering of the one or more bacteriophages; wherein the sensitivityis heritable, independent of continuing bacteriophage metabolism withinthose cells, and does not relate to the destruction of a biofilm toinduce sensitivity.
 2. (canceled)
 3. The method of claim 1 whereintesting is used to select chemical antibiotics for therapeutic use inpatients.
 4. (canceled)
 5. The method of claim 1 wherein there isinduction of sensitivity to an aminoglycoside antibiotic. 6-12.(canceled)
 13. A method to induce or improve susceptibility to anantibiotic in bacterial cells, comprising: administering to a human oranimal with a bacterial infection, a bacteriophage preparationcomprising one or more bacteriophages, and determining the ability ofsaid preparation to increase susceptibility of said cells to saidantibiotic is by in vitro antibiotic sensitivity testing in the absenceof bacteriophages using samples of bacterial cells from said infectionor from another infection by the same or a comparable bacterial strainof the same species subject to identical exposure to said bacteriophagepreparation, wherein said in vitro antibiotic testing is carried outboth before and after the exposure to said bacteriophage preparation.14. (canceled)
 15. The method according to claim 13 wherein said one ormore bacteriophages induce or improve susceptibility to anaminoglycoside antibiotic. 16-25. (canceled)
 26. The method according toclaim 1 further comprising administering at least one antibiotic. 27.The method according to claim 13 wherein the testing is used to selectchemical antibiotics for therapeutic use in a patient.
 28. The methodaccording to claim 13 further comprising administering at least oneantibiotic.
 29. A method to treat a bacterial infection in a human oranimal, comprising: testing bacteria from the bacterial infection forsusceptibility to an antibiotic, before the bacteria are exposed to abacteriophage preparation, administering the bacteriophage preparationto the human or animal, to expose the bacteria in the bacterialinfection, to the bacteriophage preparation, testing the bacteria fromthe bacterial infection for susceptibility to the antibiotic, after thebacteria are exposed to the bacteriophage preparation, and administeringthe antibiotic to the human or animal, after administering thebacteriophage preparation, wherein the bacteriophage preparationcomprises one or more bacteriophages, and susceptibility of the bacteriato the antibiotic after the bacteria are exposed to the bacteriophagepreparation, is greater than susceptibility of the bacteria to theantibiotic before the bacteria are exposed to the bacteriophagepreparation.
 30. The method according to claim 29 wherein a time periodbetween administering the bacteriophage preparation and administeringthe antibiotic is one to two days.
 31. The method according to claim 29wherein a time period between administering the bacteriophagepreparation and administering the antibiotic is one day to two months.32. The method according to claim 29, wherein the antibiotic is anaminoglycoside antibiotic.